In multiphoton microscopy, a beam of excitation light (usually pulsed infrared (IR) laser light) is brought to a focus within a specimen. Localized harmonic emission and/or multiphoton excited fluorescent emission is produced at this focus. Because of the non-linear dependency on flux intensity involved in these processes, the emitted light generated is highly localized at the point of focus and thus provides distinct information from only that point and none from the adjacent volume. In the case of two-photon fluorescent emission this signal light is approximately half the wavelength (twice the frequency) of the excitation beam. With 2nd and 3rd harmonic generation the wavelength of the signal light is exactly half or a third of the excitation wavelength. The microscopy system additionally includes a means of collecting this emitted light and projecting it onto any of a variety of photodetectors. The entire device is thus a very sensitive and spatially selective probe and detection system. Most commonly these systems also include a mechanism, such as galvanometer-controlled mirrors, to scan the focus point of excitation sequentially within the sample. Usually this motion is in the pattern of a raster scan in the x-y plane but may also include displacement along the z-axis. Using a programmable computer to combine this sequential stream of intensity measurements with the known x-y-z coordinates of the scan mechanism, a two- or three-dimensional intensity map of the specimen is created.
Conventionally, the preferred mechanism for focusing the excitation light to a point as described in the previous paragraph has been the microscope objective lens. This lens also serves as the primary “collector” of the emitted signal light. In addition, a standard microscope condenser lens or objective lens is often used behind the specimen to collect emitted signal light traveling away from the objective lens. This light can be detected separately and electronically summed with the signal light collected through the first objective lens to increase overall signal capture. For both collection pathways the conventional method of bringing the signal light to impinge on the photodetector has included some device to separate the emitted signal light from the excitation light. This can be done with a dichroic mirror for instance, a device that is spectrally selective in reflection and transmission. Additionally, some means of concentrating the signal light onto the photodetector active area is required. This is conventionally accomplished using one or more optical lens elements which collect the diverging cone of signal light from the objective lens pupil and re-image it onto the photodetector.
In a multiphoton scanning microscopy system the characteristics of the objective lens are the determining and limiting factor as to the accessible field of view and resolving capability of the entire system. The field of view is a function of the scanning system coupled with the magnification (power) of the objective lens. Lenses of lower power yield proportionally larger fields of view. The fine detail resolving power is a function of the numerical aperture (NA) of the objective lens. NA is a dimensionless number that characterizes the range of angles over which the lens can accept or emit light. The size of the finest detail that can be resolved with an objective lens is proportional to λ/NA, where λ is the wavelength of the excitation light. Thus, an objective lens of greater NA is capable of visualizing finer details than a lens of lower NA. Because of their greater acceptance angle, lenses of greater NA are also able to collect more of the signal light generated within the sample, thereby improving the sensitivity of the system. In addition to the collection efficiency of high NA lenses, a lens of lower power (i.e., wider field of view) is capable of capturing a relatively greater proportion of signal light when imaging at depth in a turbid medium.
An optical principle inherent in objective lens design is that for a given NA, the pupil diameter of the lens will vary inversely with the magnification of the objective lens. For many years, objective lens designs have evolved based upon combinations of parameters that have, for the most part, resulted in objective lens pupil diameters in the range of from about 4 mm up to about 12 mm. Most conventional multiphoton systems use objective lenses with pupil diameters in the 5 to 8 mm range. In response to an increasing interest in wider fields of view while maintaining maximum resolution, several advanced biological objective lens designs have recently been brought to the market by the major microscope manufacturers. These new objective lenses are attractive because of their combination of low magnification and high NA. These lenses, however, have pupil diameters of 16 to 17 mm and greater. Even lower power lenses (with yet larger pupils) are under development to increase further the accessible field of view.
Multiphoton imaging inherently involves very low signal return from the sample. This is especially true of biological multiphoton imaging where photodamage to the sample (caused by excessive excitation dose) is always a paramount concern. It is also a well know principle that undesirable spurious noise and dark current (current flow that is not a product of signal light) within photodetectors varies in proportion to the size of the active area of the detector. Thus, in low signal-to-noise situations (which is always the case in multiphoton imaging), there is an advantage in reducing the size of the photodetector to a practical minimum because it reduces the amount of spurious noise and dark current. Many of the best photodetectors, in terms of sensitivity, speed, and low noise, have active areas of only a few square millimeters. Additionally, photodetectors such as photomultiplier tubes have active area sensitivity profiles that rise to a peak only in an area considerably smaller than the specified full active area. With the advent and implementation of large-pupil objective lenses (and equally so with condenser lenses) an inescapable geometric difficulty arises when attempting to transfer light from a relatively large objective pupil to a relatively small photodetector active area.
A conventional, prior art multiphoton detection and scan system is illustrated schematically in FIG. 1. Here excitation light from the illumination source 1 is directed into a scanning galvanometer mechanism 2 which imparts the typical raster pattern scanning of the focused spot at the sample. The scanning beam travels through a scan lens 3, reflects off a turning mirror 5, passes through a tube lens 6, passes through a dichroic beam splitter 18, and enters the objective pupil 7. The objective lens 8 focuses the excitation light on the sample placed at sample plane 9. Signal light which may be of a wavelength different from the excitation is emitted from the specimen. A portion of the signal light which radiates upward is collected by the objective lens 8, reflects off the spectrally selective dichroic 18, passes through lens 19, and arrives at the photodetector 20 where it is converted into an electrical signal. The lens 19 and photodetector 20 together define a non-descanned detector 21. A portion of the signal light which radiates downward is collected by the condenser lens (or objective lens) 48 and passes through objective lens 49 having diameter 50, reflects off the mirror or beam splitting device 52, passes through lens 53, and arrives at the photodetector 54 where it is converted into an electrical signal. Lens 53 and the photodetector 54 together define a transmission non-descanned detector 51.
FIG. 2 illustrates a simplification of either of the prior art detection pathways of the conventional multiphoton system of FIG. 1. The light rays shown represent signal light from the sample (at plane 9) being relayed through the optical system (objective lens 8 having entrance/exit 7 and diameter 11, and focusing lens 19) to the photodetector 20. Here lens 19 is used to collect the signal light exiting the objective pupil with a diameter 11. In accord with basic optical principles, lens 19 forms an image 45 of the objective pupil 11 at the photodetector 20. This pupil image 45 at the photodetector 20 will have a diameter equal to the objective lens pupil diameter multiplied by the ratio L2/L1.
FIG. 3 illustrates the same detection path shown in FIG. 2, but with an objective lens 8 that has a pupil diameter 11 twice as large as the corresponding pupil diameter in FIG. 2. As in FIG. 2, lens 19 relays the light from the objective lens pupil plane 7 to the photodetector 20 where it now forms an image 45 having a diameter twice as large as the corresponding image produced in FIG. 2. The problem at hand is easily perceived: When the photodetector 20 in FIGS. 2 and 3 is optimized for a microscope dimensioned as in FIG. 2, that same photodetector is not optimized for a microscope dimensioned as in FIG. 3. In the device shown in FIG. 3, the larger pupil image overfills the detector active area and a significant portion of the signal is wasted. Assuming a uniform intensity across the pupil cross-section, the portion of signal lost in this case is ˜75% (based on a comparison of areas). This loss of signal is a fundamental problem when using low-power, high NA objective lenses.
FIG. 4 illustrates an additional aspect of wide-field signal collection and transfer to a photodetector that has relevance to the invention disclosed herein. The optical system depicted in FIG. 4 is the same as depicted in FIG. 3. However, in FIG. 4, a sample 46 with considerable light scattering properties is illustrated. Scattering within the sample adds randomness to the signal light before it is collected by the objective lens 8. This results in a wider emission cone 47 of signal light after the signal passes through the objective lens entrance/exit plane 7 as it travels toward the focusing lens 19. To maintain maximum detection efficiency, the focusing lens 19 must be enlarged (as shown by the dotted line around lens 19) to capture this wider cone of light. Enlarging a lens in this fashion, while holding distances L1 & L2 constant, requires that the signal light interact with greater surface curvature near the edge of lens 19. This gives rise to increasing chromatic and spherical aberrations within the pupil image 45 at the photodetector. It also results in greater entry angles of the pupil image the photodetector. All of these phenomena cause broadening of the focused image.
FIG. 5 demonstrates another challenge in wide-field signal collection and transfer to a photodetector. The optical system depicted in the upper part of the figure is the same as in FIG. 3 and serves here as a reference. The optical system in the lower part of the figure shows the objective lens 8 translated to the left a distance “X”. The leftward movement of objective lens 8 derives from a situation that arises quite commonly in multiphoton imaging work, especially when imaging large or cumbersome samples that may not be easily positioned at the ideal distance from the detection system. In these situations, the objective lens is moved toward the sample rather than the sample being moved toward the objective lens. As can be seen from the lower diagram of FIG. 5, as the objective lens 8 is moved away from focusing lens 19, the image of the objective pupil 45 also moves left away from its optimal focus on the photo-detector (or right if the objective lens 8 is moved toward focusing lens 19). In the situation illustrated in FIG. 5, where the objective pupil shifts left, the objective pupil image 45 also shifts to the left a distance “Y”. The photodetector 20 is now presented with a diverging light beam of larger diameter 58, which, as detailed below, reduces detection efficiency.
In FIG. 6 another aspect of moving the objective lens is detailed. The schematic in the upper part of the figure is identical to that of FIG. 3 and again serves as a reference. This time, in the lower schematic of FIG. 6, the objective is translated downward (in the Y axis) a distance 59. According to standard optical principles associated with the function of focusing lens 19, this downward translation of the objective lens results in the image of the objective pupil 45 moving upward a distance 60. With this translation of the pupil image 45 the signal light no longer is centered on photodetector. In fact, if the translated distance is sufficiently large, the signal might miss the photodetector entirely. Again there is a loss of efficiency.
FIGS. 7 and 8 show a more rigorous modeling of an improved variation of the conventional detection path. Here, a large aperture objective lens 48 is shown emitting signal light in a cone 54 of +/−12 degrees. Two aspheric collector lenses 49 are used in series to provide increased optical power. The increased optical power reduces the L2/L1 ratio with the net result being a smaller image of the objective pupil at a photomultiplier tube (PMT) photocathode.
The geometric situation at a photomultiplier tube (PMT) photocathode is illustrated in isolation in FIG. 8, which is a magnified view of the right-hand portion of FIG. 7. This situation applies to other minimally sized detectors as well. This method of using optical power to concentrate the signal light on the detector approaches a practical point of diminishing returns, which is evident in FIG. 8. The PMT photocathode 51 has an active width 52. Because of the optical levering involved, the signal light bundle forms a very steep cone 55 as it enters the detector structure. It can be seen from FIG. 8 that these high-angle ray paths intercept the photocathode 51 at glancing angles, many fall near the outer edges, and in some cases 53 miss the photocathode entirely or hit support structure. These last three deficiencies all represent significant signal loss.
FIG. 9 is a schematic diagram of the photocathode of a high performance and widely used commercial photodetector: the Hamamatsu R3896 photomultiplier tube. The R3896 PMT has a photocathode 56 that is 24 mm by 8 mm. The spatial sensitivity of the photocathode in the X-axis 57 and the Y-axis 58 are graphically displayed along their respective axes. The full-width half-max (FWHM) points, 60 and 61, on the sensitivity curves are shown to highlight the most sensitive area of the photocathode. As is readily apparent from the two traces 57 and 58, the spatial sensitivity of the PMT is not constant. The photocathode 56 has a “sweet spot” where it is most sensitive. To maximize the inherent capability of the R3896 PMT the signal light to be detected must impinge upon the sweet spot of the detector. Superimposed upon the diagram of the detector photocathode is an circle 59. The circle indicates the “ideal case” projection of the signal light onto the photocathode as the lens pair 49 of FIG. 7 works to form the image of the pupil of objective lens 48 of FIG. 7 at the photocathode 56. The hatched area within the circle 59 indicates the portion of the impinging signal light that falls outside of the most sensitive FWHM area of the photocathode 56 where it is either lost or detected at considerably lower efficiency. The loss of efficiency is dramatically illustrated with the photocathode of the R3896 PMT. The same geometrical challenge arises with any photo-detector of minimum size when being coupled with these large aperture objective lenses.